The present invention generally relates to diagnostic coagulation assays, and specifically, to thromboplastin reagents suitable for use in thromboplastin-based coagulation assays. The invention particularly relates, in preferred embodiments, to mammalian thromboplastin extracts, to methods for preparing such extracts, to thromboplastin reagents prepared therefrom, and to use of such reagents in prothrombin-time assays.
Thromboplastin reagents include active Tissue Factor which, when exposed to a plasma sample in the presence of calcium ions (Ca++), activates the extrinsic pathway of blood coagulation. More specifically, Tissue Factor activates Factor VII in the presence of Ca++, which in turn, activates Factor X and Factor V to initiate the formation of thrombin from prothrombin (Factor II). Thrombin is a serine endopeptidase which cleaves fibrinogen to form fibrin. Fibrin, which is soluble as a monomer, polymerizes to form a soft clot and is subsequently crosslinked to form a hard clot. The time required to form a clot upon the combination of a thromboplastin reagent and a plasma sample is a measure of the procoagulant activity of the plasma.
Diagnostic assays that employ thromboplastin reagents and are based on clotting timesxe2x80x94typically referred to as prothrombin-time (PT) assaysxe2x80x94have been used extensively for determining blood coagulation deficiencies associated with the extrinsic coagulation pathway. See Quick, Am. J. Med. Soc. 190:501 (1935). PT assays are employed, for example, for screening patients"" plasma prior to surgery. PT assays are also employed for monitoring anticoagulant treatment with pharmaceuticals, such as coumarin (Warfarin(trademark), Coumadin(trademark)), that affect the extrinsic coagulation pathway.
The primary performance criteria for thromboplastin reagents for use in PT assays are the coagulation time for a xe2x80x9cnormalxe2x80x9d plasma sample, the sensitivity of the reagent and the lot-to-lot reproducibility of the reagent. The time for coagulation of a normal plasma in a PT assay, as determined by Quick, was about 12 seconds, and the 12-second xe2x80x9cnormal PTxe2x80x9d has subsequently become an expected criteria for thromboplastin reagents in the United States. While thromboplastin reagents preferably have a PT normal time of about 12.0 seconds, times ranging from about 9.0 to about 20.0 seconds may be satisfactory under certain circumstances, and times ranging from about 9.0 to about 14.0 seconds, preferably from about 9.0 to about 13.0 seconds, are generally acceptable. The sensitivity of a thromboplastin reagent generally refers to the dynamic range in PT assay times obtained for a xe2x80x9cnormalxe2x80x9d plasma sample as compared to a xe2x80x9cstandard abnormalxe2x80x9d plasma sample. xe2x80x9cStandard abnormalxe2x80x9d plasma samples with respect to which the sensitivity of a given thromboplastin reagent is characterized include, for example, coumarinized plasma samples (e.g. plasmas treated with Coumarin(trademark) to have an International Normalized Ratio (INR) of about 3.0), and 99%+ Factor VII-deficient plasma samples. The clotting times associated with pooled normal plasmas (PNP), diluted PNP (e.g. 50%-diluted PNP), or the difference in such times are also useful parameters for evaluating thromboplastin reagents. Sensitivity may be quantitized in terms of ratios or as normalized ratios to allow for more meaningful comparison between assays employing different thromboplastin reagents.
The active Tissue Factor of a thromboplastin reagent is typically obtained by extracting homogenized mammalian tissue or an acetone powder thereof with a salt solution. Thromboplastin extractsxe2x80x94from acetone powder of rabbit brain, human brain or human placenta, for examplexe2x80x94are combined with calcium ions (Ca++), buffers and stabilizers to form an active thromboplastin reagent ready for use in a PT assay. However, thromboplastin reagents prepared in this manner from thromboplastin extracts are limited with respect to sensitivity. The insensitivity of thromboplastin-based assays is partially attributable to the presence of source-animal contaminating proteins in the thromboplastin solutionxe2x80x94especially vitamin K-dependent proteins such as Factor VII, Factor IX, Factor X, Factor II, Protein C and Protein S. These contaminating proteins stem from residual endogenous blood present with the mammalian tissue from which the thromboplastin extract is prepared.
One approach for improving the sensitivity of thromboplastin reagents involves partial absorption of vitamin K-dependent clotting factors and subsequent separation thereof from the thromboplastin extract. For example, U.S. Pat. No. 5,270,451 to Hawkins et al. discloses a method for preparing a thromboplastin extract which includes treatment with BaSO4 to adsorb the vitamin K-dependent clotting factors and removal with subsequent centrifugation. Detergents and chaotropic agents can be employed in combination with BaSO4 to enhance the absorption and/or separation of contaminants. Other methods for improving thromboplastin reagent sensitivity have focused on optimizing the relative amounts of extract, buffer, stabilizer, preservatives, Ca++ and other agents in the reagent composition. Such approaches, while providing improved thromboplastin reagent sensitivities, result in normal PT assay times that are unacceptably longer than the 12 seconds expected by medical practitioners.
Hence, there remains a need, particularly in the United States, for thromboplastin reagents which offer improved sensitivity, but which maintain the PT assay time for normal plasmas at the accepted value of about 12 seconds.
It is therefore an object of the present invention to prepare thromboplastin reagents that simultaneously meet each of the relevant performance criteria with respect to PT normal times, sensitivity and lot-to-lot reproducibility. It is also an object of the invention to prepare such reagents using commercially viable methods.
Briefly, therefore, the present invention is directed to the method for separating a plasma clotting factor from a thromboplastin solution comprising exposing a thromboplastin solution comprising Tissue Factor and a plasma clotting factor to a first surface of a semipermeable membrane and allowing the clotting factor to pass from the thromboplastin solution through the membrane and retaining the Tissue Factor in the thromboplastin solution.
The invention is further directed to a method for preparing a thromboplastin extract composition by extracting mammalian tissue with an extraction solution to form a thromboplastin extract, and separating a plasma clotting factor from the thromboplastin extract by membrane permeation. The invention is further directed to thromboplastin extracts prepared according to this method.
Additionally, the invention is further directed to a method for preparing a thromboplastin reagent by separating a plasma clotting factor from a thromboplastin extract by membrane permeation, and combining the thromboplastin extract with Ca++ ions. The invention is further directed to the thromboplastin reagents prepared according to this method.
Moreover, the invention is further directed to a method for preparing a thromboplastin reagent by exposing a feed solution that comprises a thromboplastin extract to a first surface of a semipermeable membrane, the membrane having a molecular weight cut-off ranging from about 75,000 Daltons to about 2,000,000 Daltons, collecting the exposed feed solution as a retentate, and combining the retentate with Ca++ ions. The invention is further directed to thromboplastin reagents prepared according to this method.
The present invention is further directed to a thromboplastin reagent comprising Tissue Factor extracted from mammalian tissue, and Ca++ ions. The thromboplastin reagent having procoagulant activity as determined by a prothrombin-time assay, and having a hemoglobin concentration of less than about 2.0 mg/dl.
Also, the invention is further directed to a method for determining the clotting time of a plasma sample by preparing or obtaining a thromboplastin reagent, combining the thromboplastin reagent with the plasma sample to form an assay solution, and determining the time elapsed from formation of the assay solution to detection of clot formation in the assay solution. The invention further provides a method for determining the level of a plasma clotting factor present in a test plasma by determining the clotting times of standard plasmas having various known levels of the plasma clotting factor, preparing a standard correlation between the clotting times of the standard plasmas and the level of the plasma clotting factor in the standard plasmas, determining the clotting time of the test plasma, and correlating the determined clotting time of the test plasma to the level of the plasma clotting factor based on standard deviation.
The present invention is further directed to a method for preparing a thromboplastin extract by contacting mammalian tissue with an extraction solution comprising sodium citrate at a concentration of at least about 7 mM, whereby Tissue Factor is extracted from the tissue into the extraction solution, and separating the extraction solution from the extracted tissue.
Additionally, the present invention is further directed to a method for controlling the sensitivity of a thromboplastin reagent comprising modulating the free calcium ion content of the thromboplastin reagent with a calcium buffering agent. The present invention is further directed to a method for preparing a thromboplastin reagent by preparing a thromboplastin extract comprising a calcium buffering agent, separating a plasma clotting factor from the thromboplastin extract to form a purified thromboplastin extract, and adding calcium buffering agent to the purified thromboplastin extract.
Moreover, the present invention is further directed to a method for preparing a thromboplastin extract by extracting mammalian tissue with an extraction solution comprising a phospholipase inhibitor. The present invention also provides a method for stabilizing a thromboplastin extract by combining the thromboplastin extract with a phospholipase inhibitor. The present invention is further directed to a thromboplastin extract comprising a phospholipase inhibitor.
The thromboplastin reagents of the present invention offer substantial advantages over various prior art thromboplastin reagents by simultaneously offering, in preferred embodiments: (1) normal PT times within the acceptable range of 9.0 to 13.0 seconds and (2) acceptable sensitivities with respect to each of the characterizing criteria: a dynamic range for a coumarinized plasma having a International Normalized Ratio (INR) of about 3.0 of at least about 40 seconds; a dynamic range for a 99%+ Factor-VII-deficient plasma long enough to cover the present diagnostic and/or therapeutic rangesxe2x80x94preferably of at least about 40 seconds; a dynamic range for a 50% pooled-normal plasma (versus a 100% PNP) ranging from about 3 seconds to about 6 seconds; and an International Sensitivity Index (ISI) of not more than about 1.4. As such, the thromboplastin reagents can be successfully employed in PT assays for a broad range of diagnostic applications. Moreover, the methods disclosed herein for preparing thromboplastin extracts and reagents are efficient and reproducible. As such, these methods offer commercially viable means for preparing improved thromboplastin reagents.
Other features, objects and advantages of the present invention will be in part apparent to those skilled in the art and in part pointed out hereinafter. All references cited in the instant specification are incorporated by reference. Moreover, as the patent and non-patent literature relating to the subject matter disclosed and/or claimed herein is substantial, many relevant references are available to a skilled artisan that will provide further instruction with respect to such subject matter.